The CENP-S complex is essential for the stable assembly
of outer kinetochore structure
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Citation
Amano, Miho et al. “The CENP-S complex is essential for the
stable assembly of outer kinetochore structure.” The Journal of
Cell Biology 186.2 (2009): 173-182.
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http://dx.doi.org/10.1083/jcb.200903100
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Published July 20, 2009
JCB: REPORT
The CENP-S complex is essential for the stable
assembly of outer kinetochore structure
Miho Amano,1 Aussie Suzuki,1 Tetsuya Hori,1 Chelsea Backer,2,3 Katsuya Okawa,4 Iain M. Cheeseman,2,3
and Tatsuo Fukagawa1
1
Department of Molecular Genetics, National Institute of Genetics, The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan
Whitehead Institute for Biomedical Research and 3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142
Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
2
T
he constitutive centromere-associated network
(CCAN) proteins are central to kinetochore assembly. To define the molecular architecture of this critical kinetochore network, we sought to determine the full
complement of CCAN components and to define their
relationships. This work identified a centromere protein S
(CENP-S)–containing subcomplex that includes the new
constitutive kinetochore protein CENP-X. Both CENP-S–
and CENP-X–deficient chicken DT40 cells are viable but
show abnormal mitotic behavior based on live cell analysis. Human HeLa cells depleted for CENP-X also showed
mitotic errors. The kinetochore localization of CENP-S
and -X is abolished in CENP-T– or CENP-K–deficient cells,
but reciprocal experiments using CENP-S–deficient cells
did not reveal defects in the localization of CCAN components. However, CENP-S– and CENP-X–deficient cells
show a significant reduction in the size of the kine­to­
chore outer plate. In addition, we found that intrakineto­
chore distance was increased in CENP-S– and
CENP-X–deficient cells. These results suggest that the
CENP-S complex is essential for the stable assembly of the
outer kinetochore.
Introduction
The centromere is essential for faithful chromosome segregation during mitosis. The kinetochore is assembled on centromeres to form a dynamic interface with microtubules from
the mitotic spindle (Cheeseman and Desai, 2008). To understand kinetochore structure and the mechanisms related to
chromosome segregation, it is critical to define the identity,
organization, and functional roles of the numerous kinetochore proteins.
In recent years, multiple kinetochore proteins have been identified in vertebrate cells using a combination of approaches (Foltz
et al., 2006; Izuta et al., 2006; Okada et al., 2006; Cheeseman and
Desai, 2008; Hori et al., 2008a). These studies have revealed
that a constitutive centromere-associated network (CCAN) of
proteins associates with centromeres throughout the cell cycle
and provides a platform for the formation of a functional kinetochore during mitosis. Other kinetochore proteins, including the
KNL1–Mis12 complex–Ndc80 complex (KMN) network, are
targeted to kinetochores by CCAN-containing prekinetochores
during G2 and mitosis (Cheeseman et al., 2008) to establish a
fully assembled kinetochore capable of interacting with spindle
microtubules and facilitating faithful chromosome segregation
(Cheeseman et al., 2006; DeLuca et al., 2006).
In vertebrates, 15 proteins (centromere protein C [CENP-C],
H, I, K to U, and W) have been identified as CCAN compo­
nents (Hori et al., 2008a). Based on a combination of functional
and biochemical analyses, we and others have previously demonstrated that the CCAN is divided into several subclasses
(Izuta et al., 2006; Liu et al., 2006; Okada et al., 2006; Kwon
et al., 2007; McClelland et al., 2007; Hori et al., 2008a, b).
CENP-S was originally identified as copurifying with
CENP-M or -U and was verified as a CCAN component (Foltz
et al., 2006). However, CENP-S was not detected as a stoichiometric interacting partner in the CENP-H–containing complex
in our biochemical purifications from DT40 or HeLa cells
(Okada et al., 2006). Thus, we sought to define the relationship
between CENP-S and the other CCAN subcomplexes. In this
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THE JOURNAL OF CELL BIOLOGY
4
M. Amano, A. Suzuki, and T. Hori contributed equally to this paper.
Correspondence to Tatsuo Fukagawa: tfukagaw@lab.nig.ac.jp
Abbreviations used in this paper: CCAN, constitutive centromere-associated network; IP, immunoprecipitation; KMN, KNL1–Mis12 complex–Ndc80 complex;
KO, knockout.
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Published July 20, 2009
study, we identify a new CENP-S–interacting protein and define
a function for the CENP-S–containing complex in stable outer
kinetochore assembly.
Results and discussion
CENP-X is a component of the CCAN
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JCB • VOLUME 186 • NUMBER 2 • 2009
Both CENP-S– and CENP-X–deficient
DT40 cells are viable but show defects
in mitotic progression
To understand the function of the CENP-S–CENP-X complex
and its relationship to other kinetochore components, we generated loss-of-function DT40 mutants for CENP-S and -X (Fig. S2).
We were able to isolate homozygous CENP-S/ and CENP-X/
deletions, indicating that CENP-S– and CENP-X–deficient cells
are viable. To determine the consequences of loss of CENP-S
and -X, we first examined the growth curves for CENP-S– and
CENP-X–deficient cells. Under standard growth conditions, these
knockout (KO) cells grew with similar growth rates to wild-type
cells (Fig. S3). We also observed a slight accumulation of G2/M
cells in both CENP-S– and CENP-X–deficient cell lines compared with wild-type cells by FACS analysis (Fig. S3). In addition, we observed a three- to fourfold increase in the number
of apoptotic cells in these mutants compared with control cells
based on DAPI staining with microscopic analysis, which may
reflect an accumulation of mitotic defects.
To examine the presence of any errors in mitosis, we visualized the behavior of individual CENP-S–deficient cells expressing histone H2B-RFP. Time-lapse imaging of wild-type and
CENP-S–deficient cells (Fig. 2 A) indicated the presence of some
mitotic defects in the absence of CENP-S. Although most
CENP-S–deficient cells progressed through mitosis, these cells
took a mean of 25.1 ± 6.4 min (±SD; n = 42) to progress from
prophase to anaphase, which is significantly longer than the time
observed in wild-type cells (19.3 ± 4.3 min; n = 36; P = 0.000012).
In addition, we observed that >30% of CENP-S–deficient cells
displayed anaphase bridging of chromosomes (Fig. 2 A, CENP-S
KO 1–3). These results indicate that CENP-S is important for
proper mitotic progression and chromosome segregation in DT40
cells even though it is dispensable for viability in a population.
The phenotype of CENP-S– and CENP-X–deficient cells
is similar to that of cells with KOs of CENP-O class proteins,
which are viable but show slight mitotic defects (Hori et al.,
2008b). Cells with KOs of CENP-O class proteins do not exit
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Our previous purifications using epitope-tagged CENP-H, -I, or -O
did not isolate CENP-S (Okada et al., 2006), suggesting that
CENP-S represents a distinct component of the CCAN from the
CENP-H– and CENP-O–containing complexes. To assess this
more closely, we fractionated protein extract from DT40 cells
by gel filtration chromatography and analyzed each fraction by
Western blot analysis with antibodies against CENP-O or -S.
The profile of CENP-S was clearly distinct from that of CENP-O
(Fig. 1 A), suggesting that the CENP-O–containing complex
does not contain CENP-S. To confirm the results of the gel filtration analysis, we performed immunoprecipitation (IP) experiments with cell lines in which endogenous CENP-P (a CENP-O
complex protein) or CENP-S was completely replaced with
CENP-P–Flag or CENP-S–Flag, respectively (Fig. 1 B). Mass
spectrometry indicated that the CENP-P–Flag IPs primarily
contained CENP-O, -P, -Q, -R, and -50 (U) but not CENP-S,
which is consistent with our previous analysis (Hori et al.,
2008b). Similarly, in CENP-S–Flag IPs, we did not observe
clear bands at the expected sizes for the CENP-H or -O complex
proteins on silver-stained gels (Fig. 1 B). We also confirmed the
coprecipitation using high sensitivity mass spectrometry analyses. Finally, we performed IPs with cell lines in which endogenous CENP-H or -N was completely replaced with CENP-H–Flag
or CENP-N–Flag, and we similarly did not detect CENP-S in
either IP (Fig. 1 C). These results suggest that CENP-S can be
separated from the rest of the CCAN and is distinct from the
CENP-H– or the CENP-O–containing complex. However, we
note that CENP-T was detected in CENP-S IPs using high sensitivity mass spectrometry analyses (Fig. 1 C). Consistent with
this, gel filtration chromatography of DT40 extracts revealed
two peaks of CENP-S migration, one of which co-migrates with
a CENP-T peak, although the proportion of the CENP-S that
co-migrates with CENP-T is minor (Fig. S1). CENP-T was also
detected by Western blot analysis in CENP-S IPs, but the coprecipitation efficiency of CENP-T with CENP-S is not high (Fig. S1).
Considering these data, we conclude that the CENP-S complex
is distinct from the CENP-T complex, although CENP-S may
associate weakly with the CENP-T complex.
Although we did not observe clear bands of known CCAN
proteins in CENP-S–Flag IPs, we detected a strong band with a
molecular mass of 10.5 kD (Fig. 1 B). We determined the
amino acid sequences of the peptides from this band and found
that this corresponds to the D9 (Stra13) protein (amino acid sequence from ChEST533l10 clone). We will refer to D9 (Stra13)
as CENP-X based on the results of the localization experiments
described in the next paragraph. The human homologue of
Stra13 (CENP-X) was reported as a protein whose expression is
increased upon retinoic acid induction (Scott et al., 1996). To
confirm the interaction between CENP-S and -X, we generated
DT40 cell lines in which endogenous CENP-X was completely
replaced with CENP-X–Flag and used these to conduct IPs with
anti-Flag antibodies. Western blot analysis with anti–CENP-S or
anti-Flag antibodies indicated clear coprecipitation of CENP-S
and -X (Fig. 1 D). To determine whether the interaction between
CENP-S and -X is conserved in humans, we conducted similar
IPs from HeLa cell lines stably expressing GFPLAP–CENP-S or
GFPLAP–CENP-T. Mass spectrometric analysis revealed the presence of CENP-X (Stra13; NCBI Protein database accession no.
NP_659435) in CENP-S IPs but not in CENP-T IPs (Fig. 1 C).
To investigate the localization of CENP-X, we generated a
DT40 cell line in which endogenous CENP-X was completely
replaced with CENP-X–GFP. Colocalization with anti–CENP-C
antibodies demonstrated that CENP-X–GFP localizes to centromeres throughout the cell cycle (Fig. 1 E). We also confirmed the
colocalization with anti-Flag antibodies in cells in which endogenous CENP-X was completely replaced by CENP-X–Flag
(Fig. S1). Based on the copurification with CENP-S and its constitutive localization to centromeres, we conclude that CENP-X
is a new component of the CCAN.
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Figure 1. Identification of the CENP-S–associated protein CENP-X. (A) Immunoblots of DT40 cell extracts fractionated on a Superose 6 gel filtration column
using antibodies against anti–CENP-O or –CENP-S. Signal intensities are plotted in the graph, and peak fractions are indicated by arrowheads. (B) SDSPAGE of proteins isolated by IP with anti-Flag antibodies using cells in which expression of CENP-P or -S was replaced with CENP-P–Flag or CENP-S–Flag,
respectively. Wild-type (WT) DT40 cells were also used for IP with anti-Flag antibodies as a control. (C) High sensitivity mass spectrometric analysis of
the purifications of chicken and human centromere proteins indicating the percentage sequence coverage for each polypeptide. (D) Co-IP of CENP-S with
CENP-X. Immunoprecipitates of CENP-X–expressing and wild-type cells with anti-Flag antibodies were separated by SDS-PAGE and analyzed by Western
blotting with anti-Flag or –CENP-S antibodies. (E) Localization of GFP-tagged CENP-X throughout the cell cycle in DT40 cells. Centromeres were costained
with anti–CENP-C antibodies. M, molecular mass marker. Bar, 10 µm.
CHARACTERIZATION OF THE CENP-S COMPLEX • Amano et al.
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mitosis after release from a nocodazole block (Minoshima
et al., 2005). Therefore, we tested the ability of CENP-S–deficient
cells to recover from spindle damage (Fig. S3 C). We treated cells
with nocodazole for 12 h and then removed the drug and examined
the entry of the cells into interphase. Wild-type cells progressed
to G1 phase immediately, and <10% were mitotic 4 h after release from the nocodazole block. In contrast, 60% of CENP-50
(U) (a CENP-O class protein)–deficient cells were mitotic 4 h
after release from the nocodazole block, as reported previously
(Minoshima et al., 2005). In CENP-S–deficient cells, 15% of
cells at 4 h were mitotic after release from the nocodazole block.
Although this suggests a slight defect in the recovery from spindle damage in CENP-S–deficient cells compared with wild-type
cells, the defect is minor compared with CENP-50 (U)–deficient
cells. These results suggest that CENP-S–CENP-X function is
distinct from the CENP-O class of proteins.
Finally, we also analyzed the mitotic behavior after depletion of human CENP-X by siRNA-based knockdown in HeLa
cells (Fig. 2 B). In contrast to DT40 cells, CENP-X RNAi in
HeLa cells resulted in numerous mitotic defects. Although we
observed a small subset of CENP-X–depleted cells in which
chromosomes were largely aligned at a metaphase plate, numerous instances of moderately and highly misaligned chromosomes were observed in these cells (Fig. 2 B). This strong defect
in chromosome alignment is qualitatively similar to our previous analyses of CENP-K– and CENP-W–depleted HeLa cells
(Okada et al., 2006; Hori et al., 2008a). However, in contrast to
CENP-K and -W depletions, we did observe some examples of
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cells that had apparently entered anaphase based on the presence of separated sister kinetochores.
To determine whether CENP-X has overlapping functions
with other CCAN proteins, we also conducted double depletions between CENP-X and CENP-Q, -K, or -W by RNAi. This
strategy previously demonstrated a synergy between CENP-K
and human KNL1 for assembly of the human kinetochore
(Cheeseman et al. 2008). However, double CENP-X/CENP-Q–,
CENP-X/CENP-K–, and CENP-X/CENP-W–depleted cells did
not show synergistic phenotypes compared with the singly depleted cells (unpublished data). In total, these results suggest
that depletion of human CENP-X causes defects that are distinct from those associated with other CCAN components and
suggests that CENP-X plays a critical role in proper chromosome segregation.
Kinetochore localization of the
CENP-S–CENP-X complex occurs
downstream of the CENP-H complex
but is distinct from the CENP-O complex
To define the relationship of CENP-S and -X to the other components of the CCAN, we next conducted localization experiments. We found that CENP-X localization was abolished in
CENP-S–deficient cells and that CENP-S localization was
eliminated in CENP-X–deficient cells (Fig. 3 A). We also observed that CENP-X RNAi in human cells eliminated GFP–
CENP-S kinetochore localization (Fig. S3 D). In combination
with the biochemical purifications described in the previous
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Figure 2. Both CENP-S– and CENP-X–deficient DT40 cells are viable but show defects in mitotic progression. (A, left) Dynamics of chromosomes in wild-type
(WT) or CENP-S–deficient cells visualized by time-lapse observation of living cells. Selected images of chromosomes from prophase to anaphase in these
cells are shown. (right) Quantification of the time for progression from prophase to anaphase in wild-type and CENP-S–deficient cells as determined by timelapse microscopy of living cells. (B) Chromosome morphology and -tubulin staining (green) in human HeLa cells after siRNA-based knockdown for CENP-X.
Human anticentromere antibodies (ACA) were used to detect the position of centromeres (red), and DNA (blue) was stained with Hoechst. Bars, 10 µm.
Published July 20, 2009
Figure 3. Kinetochore localization of the CENP-S–
CENP-X complex occurs downstream of the CENP-H
complex but is distinct from the CENP-O complex.
(A) CENP-X localization in CENP-S–deficient cells
(CENP-S OFF) and CENP-S localization in CENP-X–
deficient cells (CENP-X OFF). WT, wild type. (B) CENP-S
localization in CENP-K– or CENP-T–deficient cells
(CENP-K OFF or CENP-T OFF, respectively). (C) Immuno­
fluorescence analysis in wild-type DT40 and CENP-50–
and CENP-X–deficient cells with the indicated antibodies
(green). DNA was counterstained with DAPI (blue).
(D) Imaging of human HeLa cells stably expres­sing
GFP-tagged CENP-H after siRNA-based knockdown
for control or CENP-X. Bars, 10 µm.
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section, these results support a model in which CENP-S and -X
form a closely associated complex.
We next examined the kinetochore localization of CENP-S
in CENP-K (a component of the CENP-H–containing complex)– or CENP-T–deficient cells. The kinetochore localization
of CENP-S was eliminated in both CENP-K– and CENP-T–
deficient cells (Fig. 3 B). We also examined the kinetochore
localization of representative CCAN components, including
CENP-C, -T, -K, -50 (U), and -S in CENP-X– and CENP-50 (U)–
deficient cells. Our observations indicate that the localization of
the tested CCAN proteins did not change in CENP-X– and
CENP-50 (U)–deficient cells (Fig. 3 C).
The phenotypes of the CENP-S– and CENP-X–deficient
cells are distinct from those of cells with KO of the CENP-O
complex proteins based on the recovery from spindle damage
assay (Fig. S3 C). Consistent with these distinct functional roles,
CHARACTERIZATION OF THE CENP-S COMPLEX • Amano et al.
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Published July 20, 2009
we observed clear CENP-S signals in CENP-50 (U)–deficient
cells (Fig. 3 C), indicating that the kinetochore localization and
function of the CENP-S–CENP-X complex is distinct from the
CENP-O complex. We have previously shown that CENP-S
localization does not change in CENP-C–deficient cells (Hori et al.,
2008a), suggesting that CENP-C has distinct function from the
CENP-S–CENP-X complex. We also confirmed that the localization of human CENP-H and -O is not affected in human cells
depleted for CENP-X (Fig. 3 D and not depicted). In total, these
localization experiments demonstrate that kinetochore targeting of
the CENP-S–CENP-X complex occurs downstream of the CENP-H
complex and is distinct from CENP-C and the CENP-O complex.
Kinetochore outer plates of
CENP-S–deficient cells are smaller than
those of wild-type cells
Intrakinetochore distance is increased in
CENP-S– and CENP-X–deficient cells in the
absence of tension
During our analysis of outer plate structure by EM, we observed
that the distance between the outer plate and electron-dense
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JCB • VOLUME 186 • NUMBER 2 • 2009
CENP-S– and CENP-X–deficient cells
display reduced localization of outer
kinetochore proteins
The EM analysis suggests that there are defects in the proper
formation of a functional kinetochore outer plate in CENP-S–
deficient cells. Therefore, we next analyzed the localization of
outer kinetochore proteins in CENP-S– or CENP-X–deficient
cells. The KMN network is located at the outer plate (DeLuca
et al., 2005) and has a central role in forming kinetochore–
microtubule attachments (Cheeseman et al., 2006). Although
the KMN network subunits KNL1, Mis12, and Ndc80 all localized to kinetochores in CENP-S– and CENP-X–deficient DT40
cells, we found that the signal intensities of Ndc80 (80% relative to control; Fig. 5 A) and KNL1 (not depicted) were reduced
in CENP-S– and CENP-X–deficient cells relative to controls. In
contrast, we did not detect significant differences of Mis12 signals between the control and mutant cells.
We also conducted similar experiments to determine whether
CENP-X is required for outer kinetochore assembly in human cells
(Fig. 5 B). We observed a strong reduction in the levels of human
KNL1 (35 ± 38% relative to control) and Ndc80 (53 ± 11% relative
to control) at kinetochores, but the level of Dsn1 (a Mis12 complex
protein) was not changed. In total, these results demonstrate that
the assembly of the outer kinetochore components KNL1 and
Ndc80 is compromised in CENP-S– and CENP-X–deficient cells,
which causes reduced outer plate formation.
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Although we did not observe an apparent reduction in the kinetochore localization of established CCAN proteins in CENP-S– or
CENP-X–deficient cells (Fig. 3), it is possible that CENP-S and
-X play a role in controlling outer kinetochore assembly. To determine whether CENP-S or -X play a global role in controlling
the assembly of non-CCAN proteins, we examined the structural
morphology of the kinetochore outer plate by EM. For these experiments, we imaged 30–40 170-nm-thick serial sections for individual mitotic cells treated with nocodazole. In both CENP-S–
deficient cells and wild-type DT40 cells, we observed similar
numbers of clear electron-dense kinetochore outer plates (Fig. 4,
A and B). We also observed a similar number (26.6 ± 8.8) of outer
plates in CENP-50 (U)–deficient cells. In contrast, we observed
that CENP-H deficiency causes a severe reduction in the number
of visible outer plates (unpublished data), as previously shown
for CENP-T deficiency (Hori et al., 2008a).
Although we did not detect differences in the numbers of
outer plates between CENP-S–deficient cells and wild-type DT40
cells, we found that the plate length of CENP-S–deficient cells
was shorter than that of wild-type cells. As shown in Fig. 4 C, the
length of outer kinetochore plates varied even in individual cells.
In wild-type cells, we observed a main peak of plate length of
200 nm and a shoulder peak of 300 nm, with a mean plate
length of 227 nm (±70.5 nm; Fig. 4 C). In CENP-S–deficient
cells, we observed a main peak of 150 nm and a shoulder peak
of 240 nm, with a mean size of 185 nm (±61.7 nm; Fig. 4 C).
The plate length of CENP-50 (U)–deficient cells (261 ± 148.5 nm)
was not significantly different from that of wild-type cells.
These data suggest that CENP-S is involved in the formation of
a functional kinetochore outer plate, which is essential for
kinetochore–microtubule attachment and faithful mitotic progression. Liu et al. (2006) have previously reported that outer plate
morphology is altered after treatment of siRNA against CENP-I.
The contribution of CENP-S to the formation of a functional
kinetochore may function coordinately with the CENP-I pathway.
chromatin region in CENP-S–deficient cells was longer than
that in wild-type cells (Fig. 4 A). Therefore, we measured the
distance in 150 kinetochores from our EM analysis for both
CENP-S–deficient and wild-type cells and found that the distance in CENP-S–deficient cells (29.9 nm) is significantly longer
than that in wild-type cells (23.9 nm; Fig. 4 D). The distance in
CENP-50 (U)–deficient cells was 26.3 nm, which is slightly
longer than that in wild-type cells but shorter than that in CENP-S–
deficient cells. Recent studies suggested that the condensin
complex regulates inter- and intrakinetochore distances under
tension (Maresca and Salmon, 2009; Uchida et al., 2009). However, inter- and intrakinetochore distances are not changed in
condensin-null DT40 cells in the absence of tension (Ribeiro et al.,
2009), which suggests that there is a mechanism to prevent the
kinetochore from stretching in the absence of external force.
Our EM analysis was performed after treatment with nocodazole,
which eliminates kinetochore–microtubule attachments and spindle
forces. To confirm the EM observations, we stained chromosomes with anti–CENP-T (an inner kinetochore protein) or antiNdc80 (Hec1; an outer kinetochore protein) antibodies after
nocodazole treatment and measured the distances between sister kinetochores in CENP-S– and CENP-X–deficient and wildtype cells. The distance between Ndc80 at two paired sister
kinetochores was increased in CENP-S–CENP-X–deficient cells.
In contrast, the distance between CENP-T between two paired
inner kinetochores was not changed (Fig. 4 E). These results indicate that the intrakinetochore distance is increased in both
CENP-S and –CENP-X–deficient cells and that this occurs
structurally between the inner kinetochore protein CENP-T and
the outer kinetochore protein Ndc80.
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Figure 4. The length of the outer plate and the distance of the interkinetochore in CENP-S–deficient cells. (A) Electron micrograph showing a typical
image of the kinetochore outer plate in wild-type (1 and 1) or CENP-S–deficient cells (2 and 2). (B) Numbers of identifiable outer plates per cell (mean
± SD). Approximately 35 serial sections were made for each cell, and the numbers of outer plates were counted. (C) Distribution of the outer plate length
in wild-type or CENP-S–deficient cells. In both cell lines, two peaks with distribution were observed. The two peaks in the distribution of the plate length are
likely caused by differences in sample orientation. As the orientation of some cells is not plane of the section, the length of plates varies depending on the
angle of the section against a chromosome. (D) Distance between outer plate and electron-dense chromatin region in wild-type or CENP-S–deficient cells.
(E) Distances between Ndc80 and Ndc80 or CENP-T and CENP-T of control or CENP-S– or CENP-X–deficient metaphase cells in the absence of micro­
tubules. Arrows indicate sister kinetochore pairs. (D and E) Error bars represent SD. Bars: (A) 200 nm; (E) 5 µm.
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Figure 5. Localization of outer plate proteins in CENP-S– or CENP-X–deficient cells. (A) Immunofluorescence analysis in wild-type (WT) DT40 or CENP-S–
or CENP-X–deficient cells with anti-Ndc80 and -Mis12 antibodies. Signal intensities at each kinetochore were measured in these cells. Error bars represent
SD. (B) Immunofluorescence analysis with anti-Ndc80, -KNL1, or -Dsn1 antibodies in HeLa cells treated with control or CENP-X siRNAs. The numbers in
the micrographs are relative signal intensities of kinetochore signals. (C) A model for the assembly of kinetochore proteins at mitosis. The CENP-S–CENP-X
complex is essential for the stable assembly of outer kinetochore proteins. In addition, the CENP-S–CENP-X complex generates a discrete CCAN structure
to prevent the kinetochore from overstretching (left). In CENP-S– and CENP-X–deficient cells, the amount of KNL1 and Ndc80 at kinetochores is reduced,
and CCAN structure is less tight, which causes kinetochore to be overstretched (right). Bars, 10 µm.
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JCB • VOLUME 186 • NUMBER 2 • 2009
Published July 20, 2009
A functional role for the CENP-S–CENP-X
complex in the assembly of a
kinetochore structure
Materials and methods
Molecular biology, cell culture, and transfection
Chicken target disruption constructs for each gene were generated such
that genomic fragments encoding several exons were replaced with drug
resistance cassettes under control of the -actin promoter. Target constructs
were transfected with a Gene Pulser II electroporator (Bio-Rad Laboratories).
All DT40 cells were cultured at 38°C in Dulbecco’s modified medium supplemented with 10% fetal calf serum, 1% chicken serum, 2-mercaptoethanol,
penicillin, and streptomycin. To repress the expression of the tetracyclineresponsive transgenes, tetracycline (Sigma-Aldrich) was added to the culture medium to a final concentration of 2 µg/ml. To deplete human CENP-X,
a pool of four siRNAs was obtained from Thermo Fisher Scientific (5-CACCUGCACUUCAAGGAUG-3, 5-GACCAAAGAAGCAGCAGUC-3,
5-CGCAGCUGCUCCUGGACUU-3, and 5-AC­G­UGG­ACCA­GCUG­GA­
GAA-3) and transfected into cells using Oligofectamine (Invitrogen) according to the manufacturer’s guidelines.
Biochemical analysis
DT40 whole cell extracts were suspended in lysis buffer (50 mM Na phosphate, pH 8.0, 0.3 M NaCl, 0.1% NP-40, 5 mM -mercaptoethanol, complete protease inhibitor [Roche], and 20 U/ml DNase I [Takara]) and were
centrifuged at 20,000 g for 10 min at 4°C. The soluble supernatant was
fractionated on a Superose 6 gel filtration column (GE Healthcare) in 50 mM
Na phosphate, pH 8.0, 0.3 M NaCl, 0.3 M imidazole, 0.1% NP-40, and
1 mM -mercaptoethanol at 4°C.
For IP experiments, DT40 cells expressing Flag-tagged proteins were
lysed in 5 ml of lysis buffer and centrifuged at 20,000 g for 10 min at 4°C.
Anti-Flag M2 beads (Sigma-Aldrich) were incubated with the supernatant
fraction for 2 h at 4°C, washed with lysis buffer, and eluted with lysis buffer
in the presence of 3× Flag peptide (Sigma-Aldrich).
Immunofluorescence and image acquisition
Cells were collected onto slides with a cytocentrifuge and fixed in 3%
paraformaldehyde in PBS for 15 min at room temperature or in 100%
Living cell observation
For live cell imaging, a histone H2B-RFP plasmid was transfected into
CENP-S–deficient cells to visualize chromosomes. Fluorescently stained living cells were observed with an IX71 inverted microscope with an oil
immersion objective lens (Plan-Apochromat 60× NA 1.40) in a temperaturecontrolled box to keep the temperature at 38°C. Time-lapse images were
recorded at 3-min intervals with an exposure time of 0.2–0.3 s using a
CoolSNAP HQ2 camera. Subsequent analysis and processing of images
were performed using IPLab software (Signal Analytics).
EM
DT40 cells were treated with 500 ng/ml nocodazole for 3 h and fixed
in 2.5% glutaraldehyde and 0.15% tannic acid in the 0.1 M Na cacodylate buffer for 1 h. Postfixation was performed in 2% OsO4 for 1 h on
ice. The cells were dehydrated in ethanol and then infiltrated with Epon
812. Polymerization was performed at 60°C for 48 h. Serial sections
were cut with an ultramicrotome (Leica) equipped with a diamond knife
(Diatome), and sections were stained with uranyl acetate and lead
citrate and examined in an electron microscope (JEM1010; JEOL). The
contrast of each image was normalized with the brightness of the membrane structure.
Online supplemental material
Fig. S1 shows the relation of the CENP-S–CENP-X complex with the CENP-T–
CENP-W complex. Fig. S2 indicates the strategy for generating KO cell
lines for CENP-S and -X. Fig. S3 shows the phenotype for CENP-S– and
CENP-X–deficient cells. Online supplemental material is available at
http://www.jcb.org/cgi/content/full/jcb.200903100/DC1.
Downloaded from jcb.rupress.org on January 11, 2010
Based on phenotype analysis of CENP-S– and CENP-X–deficient
cells and our previous work on the function and organization of the
other CCAN components (Okada et al., 2006; Hori et al.,
2008a, b), we propose a model in which the CENP-S–CENP-X
complex is essential for the stable assembly of outer kinetochore
proteins (Fig. 5 C). Although CENP-S has a potential histone-fold
domain, the significance of this domain to the function of CENP-S–
CENP-X complex is unclear. As the data in this study indicate, the
CENP-S–CENP-X complex functions downstream of other components of the CCAN, and depletion of the CENP-S–CENP-X
complex does not affect the localization of other CCAN proteins.
Thus, the CENP-S–CENP-X complex may directly allow the outer
kinetochore to form efficiently and stably on the CCAN platform.
We also found that the intrakinetochore distance was increased in CENP-S– and CENP-X–deficient cells in the absence of
tension (Fig. 4 E). Combined with our EM analysis, the inner
kinetochore structure appears to be stretched in CENP-S–deficient
cells (Fig. 5 C, right). To our knowledge, these are the first proteins
that have been identified with a role in kinetochore assembly related to the structure of the intrakinetochore distance in the absence
of tension. Although this function may not be essential under all
circumstances, as is seen for the null phenotype in DT40 cells, the
ability to assemble a coherent kinetochore structure capable of
resisting the force from the spindle microtubules and preventing
merotelic attachment to opposing spindle poles is critical to the
integrity of this important cell division structure.
methanol for 15 min at 20°C, permeabilized in 0.5% NP-40 in PBS for
10 min at room temperature, rinsed three times in 0.5% BSA, and incubated for 1 h at 37°C with primary antibody. Binding of primary antibody
was then detected with Cy3- or FITC-conjugated goat anti–rabbit IgG (Jackson
ImmunoResearch Laboratories) diluted to an appropriate concentration in
PBS/0.5% BSA. Affinity-purified rabbit polyclonal antibodies were used
against recombinant chicken CENP-C (Fukagawa et al., 1999), CENP-H
(Fukagawa et al., 2001), CENP-K (Okada et al., 2006), CENP-50
(Minoshima et al., 2005), CENP-T (Hori et al., 2008a), and CENP-S.
Other antibodies used in this study were described previously (Nishihashi
et al., 2002; Hori et al., 2003; Regnier et al., 2005). Chromosomes and
nuclei were counterstained with DAPI at 0.2 µg/ml in Vectashield antifade
(Vector Laboratories). Immunofluorescence images were collected with a
cooled EM charge-coupled device camera (Quntem; Roper Scientific)
mounted on an inverted microscope (IX71; Olympus) with a 100× NA
1.40 objective lens together with a filter wheel at room temperature. Subsequent analysis and processing of images were performed using MetaMorph software (Roper Scientific). Immunofluorescent human cells were
imaged on a Deltavision Core system (Applied Precision, LLC) equipped
with a camera (CoolSNAP HQ2; Photometrics). All images were scaled
and processed identically.
We are very grateful to K. Suzuki, M. Takahashi, and K. Kita for technical
assistance. We also thank E. Suzuki for advice on EM experiments.
This work was supported by Grants-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Technology of Japan to
T. Fukagawa and awards from the Richard and Susan Smith Family Foundation, the Massachusetts Life Sciences Center, and the Searle Scholars Program
to I.M. Cheeseman.
Submitted: 18 March 2009
Accepted: 24 June 2009
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